Abstract The central hypothesis of this proposal is that continued production of aggregation prone proteins leads to age onset proteotoxicity and eventually disease. Two areas will be studied using proteomics technology. The first is to identify the proteins involved in disassembly/proteolysis of protein aggregates. By using newly developed assays in the Kelly laboratory, disassembly and proteolysis activities will be followed over the course of biochemical enrichment. Proteomics will be used during the enrichment process to follow and assess the course of enrichment and then finally to identify the proteins responsible for the activity. Detoxification activities are under the control of the transcription factors HSF-1 and DAF-16. We will identify the proteins whose expression levels are under the control of these transcription factors by using quantitative proteomics when the levels of HSF-1 and DAF-16 are reduced by RNAi. The methods, techniques, and experience are available in the proteomics core to conduct these analyses. The core will employ large-scale protein identification technology developed in our lab called MudPIT (Multi-dimensional Protein Identification Technology). To perform MudPIT experiments we have several different types of mass spectrometers that can be used including LTQ-Orbitrap, LTQ and LTQ-ETD (Electron Transfer Dissociation) systems. Each instrument has particular strengths that can be applied to these projects. The LTQ-Orbitrap produces high resolution and high mass accuracy data that is good for confident protein identifications, the discovery of post translational modifications, and accurate quantitation. The LTQ is a slightly more sensitive and faster scanning instrument than the hybrid and can be used when sensitivity and sequence coverage are important. The LTQ-ETD system uses a new dissociation technique that is good for fragmenting large polypeptides. This particular instrument may have an advantage when attempting to identify proteins from aggregates or proteins that do not digest well.